Transmission of Vibrio cholerae is antagonized by lytic phage and entry into the aquatic environment.
Bottom Line: Phage did not affect colonization immediately after shedding from the patients because the phage titer was too low.Phage had an undetectable impact on this adaptation.Taken together, the rise of ABNC cells and lytic phage blocked transmission.
Affiliation: Howard Hughes Medical Institute, USA.
Cholera outbreaks are proposed to propagate in explosive cycles powered by hyperinfectious Vibrio cholerae and quenched by lytic vibriophage. However, studies to elucidate how these factors affect transmission are lacking because the field experiments are almost intractable. One reason for this is that V. cholerae loses the ability to culture upon transfer to pond water. This phenotype is called the active but non-culturable state (ABNC; an alternative term is viable but non-culturable) because these cells maintain the capacity for metabolic activity. ABNC bacteria may serve as the environmental reservoir for outbreaks but rigorous animal studies to test this hypothesis have not been conducted. In this project, we wanted to determine the relevance of ABNC cells to transmission as well as the impact lytic phage have on V. cholerae as the bacteria enter the ABNC state. Rice-water stool that naturally harbored lytic phage or in vitro derived V. cholerae were incubated in a pond microcosm, and the culturability, infectious dose, and transcriptome were assayed over 24 h. The data show that the major contributors to infection are culturable V. cholerae and not ABNC cells. Phage did not affect colonization immediately after shedding from the patients because the phage titer was too low. However, V. cholerae failed to colonize the small intestine after 24 h of incubation in pond water-the point when the phage and ABNC cell titers were highest. The transcriptional analysis traced the transformation into the non-infectious ABNC state and supports models for the adaptation to nutrient poor aquatic environments. Phage had an undetectable impact on this adaptation. Taken together, the rise of ABNC cells and lytic phage blocked transmission. Thus, there is a fitness advantage if V. cholerae can make a rapid transfer to the next host before these negative selective pressures compound in the aquatic environment.
Related in: MedlinePlus
Mentions: Because ABNC V. cholerae have low infectivity, yet represent the predominant state of the bacteria after 5 h of incubation in the pond microcosm, we measured possible transcriptional changes during this transition. The goal was to ascertain whether the bacteria were adapting to the nutrient poor conditions in a manner dependent or independent of their source of origin (patient or in vitro). Samples for the microarray fell into six biological groups: patient derived samples (EN159, EN182) incubated in the pond microcosm for 0, 5 and 24 h (designated T0P*, T5P* and T24P*, respectively; Fig. 6A) and the paired in vitro derived isolates incubated in the pond for 0, 5 and 24 h (designated T0I, T5I and T24I, respectively; Fig. 6A). Both patient (EN159, EN182) samples harbored phage, which is indicated with an asterisk. Two additional patient samples that did not harbor phage (EN124, EN150) were included as controls for transcriptional changes induced by phage (designated T0P, T5P and T24P, respectively; Fig. 7A). The patient samples EN174 and EN191 and in vitro samples EN124 and EN150 were excluded because of insufficient material for microarray analysis. The qRT-PCR validation is provided in Table S2. Cluster analyses in Fig. 6 and Fig. 7 isolated key expression patterns; genes within these groupings are described by biological function in Tables S3, S4, S5, S6, S7, S8, S9 and S10. A complete list of all fold changes is available in Table S10.